Many immune responses occur only if a CD4 T lymphocyte recognizes foreign antigen in association with a self class II molecule of the major histocompatibility complex (MHC). Antigen must be processed for presentation by class II molecules and this generally involves endocytosis of antigen into an acidic compartment where newly synthesized class II molecules have been transported. The foreign peptide/MHC class II complexes recognized by CD4 T cells are thus formed intracellularly. The aim of this project is to define the potential sources of antigen for presentation by class II molecules, and to determine how class II molecules are transported to and from antigen-processing compartments. The classical pathway for presentation of exogenous antigen requires newly synthesized class II molecules associated with the invariant chain, and requires HLA-DM, a class II-related molecule encoded in the MHC. However, it was demonstrated here that several processing pathways exist for antigen presentation by class II molecules. First, in contrast to the classical pathway, presentation of an influenza virus antigen was independent of protein synthesis, of the invariant chain, and of HLA-DM. Evidence was obtained that mature class II molecules at the cell surface internalize and recycle to the cell surface. In addition, cytosolic antigen can also be presented to class II-restricted T cells in a manner clearly distinct from the class I pathway, as (i) the invariant chain blocks the presentation of cytosolic peptides by class II molecules and (ii) the transporter for antigen presentation is not required whereas the class II region, including HLA-DM, is. Transport of newly synthesized class II molecules to endosomes is mediated by the invariant chain (Ii), but the intracellular transport route is not known. Using a novel assay based on labeling with galactose and trimming of terminal sialic acid residues with neuraminidase, it has been demonstrated that a large population of class II-Ii complexes reach the cell surface or early endosomes prior to their delivery to late endosomal compartments. The only precedent for retrieval to the cell surface of molecules in late endocytic compartments involves transport through the trans-Golgi network. However, evidence was obtained that class II molecules are transported back to the cell surface by a novel pathway distinct from retrieval through the trans-Golgi network.